Lipoprotein analysis by differential charged-particle mobility

ABSTRACT

The invention provides methods of preparation of lipoproteins from a biological sample, including HDL, LDL, Lp(a), IDL, and VLDL, for diagnostic purposes utilizing differential charged particle mobility analysis methods. Further provided are methods for analyzing the size distribution of lipoproteins by differential charged particle mobility, which lipoproteins are prepared by methods of the invention. Further provided are methods for assessing lipid-related health risk, cardiovascular condition, risk of cardiovascular disease, and responsiveness to a therapeutic intervention, which methods utilize lipoprotein size distributions determined by methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/711,778, filed Sep. 21, 2017, now abandoned, which is a Divisional ofU.S. application Ser. No. 14/726,802, filed Jun. 1, 2015, now U.S. Pat.No. 9,791,464, which is a Continuation of U.S. application Ser. No.14/226,089, filed Mar. 26, 2014, now U.S. Pat. No. 9,046,539, which is aContinuation of U.S. application Ser. No. 13/589,404, filed Aug. 20,2012, now U.S. Pat. No. 8,709,818, which is a Continuation of U.S.application Ser. No. 11/760,672, filed Jun. 8, 2007, now U.S. Pat. No.8,247,235, the entire contents of each are incorporated herein byreference in their entireties for any and all purposes.

FIELD OF THE INVENTION

The present invention generally relates to the fields of particle sizeanalysis and analyses of biological particles including lipoproteins fordiagnostic purposes utilizing ion mobility measurement devices andmethods. The present invention further provides methods for purificationand isolation of biomolecules including, without limitation,lipoproteins and biological complexes containing lipoproteins.

BACKGROUND OF THE INVENTION

The following description is provided solely to assist the understandingof the present invention. None of the references cited or informationprovided is admitted to be prior art to the present invention. Allpatents and other references cited in the specification are incorporatedby reference in their entireties, including any tables and figures, tothe same extent as if each reference had been incorporated by referencein its entirety individually.

Cardiovascular disease is the leading cause of death in the UnitedStates. The most commonly used and accepted methods for determining riskof future heart disease include determining serum levels of cholesteroland lipoproteins, in addition to patient demographics and currenthealth. The terms “lipoprotein” and “lipoprotein particle” as well knownin the art refer to particles obtained from mammalian blood whichinclude apolipoproteins biologically assembled with noncovalent bonds topackage for example, without limitation, cholesterol and other lipids.Lipoproteins preferably refer to biological particles having a sizerange of 7 to 120 nm, and include VLDL (very low density lipoproteins),IDL (intermediate density lipoproteins), LDL (low density lipoproteins),Lp(a) [lipoprotein (a)], HDL (high density lipoproteins) andchylomicrons as defined herein. “Biological particle” refers to amaterial having a non-covalently bound assembly of molecules derivedfrom a living source. Examples without limitation of biologicalparticles are lipoproteins assembled for example from apolipoproteinsand lipids; viral components assembled from non-covalently bound coatproteins and glycoproteins; immune complexes assembled from antibodiesand their cognate antigens, and the like. Lipoprotein density can bedetermined directly by a variety of physical biochemical methods wellknown in the art, including without limitation equilibrium densityultracentrifugation and analytic ultracentrifugation. Lipoproteindensity may also be determined indirectly based on particle size and aknown relationship between particle size and density. Lipoprotein sizemay be determined by a variety of biochemical methods well known in theart including, without limitation, methods described herein. The term“apolipoprotein” refers to lipid-binding proteins which constitutelipoproteins. Apoliproteins are classified in five major classes: Apo A,Apo B, Apo C, Apo D, and Apo E, as known in the art. There are wellestablished recommendations for cut-off values for biochemical markers,for example without limitation cholesterol and lipoprotein levels, fordetermining risk. The terms “marker,” “biochemical marker” and liketerms refer to naturally occurring biomolecules (or derivatives thereof)with known correlations to a disease or condition. However, cholesteroland lipoprotein measurements are clearly not the whole story because asmany as 50% of people who are at risk for premature heart disease arecurrently not encompassed by the ATP III guidelines (i.e., AdultTreatment Panel III guidelines issued by the National CholesterolEducation Program and the National Heart, Lung and Blood Institute).

Methods to measure lipoprotein and other lipids in the blood include,for example without limitation, evaluation of fasting total cholesterol,triglyceride, HDL (high density lipoprotein) and/or LDL (low densitylipoprotein) cholesterol concentrations. Currently, the most widely usedmethod for measuring LDL cholesterol is the indirect Friedewald method(Friedewald, et al., Clin. Chem., 1972, 18:499-502). The Friedewaldassay method requires three steps: 1) determination of plasmatriglyceride (TG) and total cholesterol (TC), 2) precipitation of VLDL(very low density lipoprotein) and LDL, and 3) quantitation of HDLcholesterol (HDLC). Using an estimate for VLDLC as one-fifth of plasmatriglycerides, the LDL cholesterol concentration (LDLC) is calculated bythe formula: LDLC=TC-(HDLC+VLDLC). While generally useful, theFriedewald method is of limited accuracy in certain cases. For example,errors can occur in any of the three steps, in part because this methodrequires that different procedures be used in each step. Furthermore,the Friedewald method is to a degree indirect, as it presumes that VLDLCconcentration is one-fifth that of plasma triglycerides. Accordingly,when the VLDL of some patients deviates from this ratio, furtherinaccuracies occur.

Another method for evaluating blood lipoproteins contemplatesmeasurement of lipoprotein size and density. The size distribution oflipoproteins varies among individuals due to both genetic and nongeneticinfluences. The diameters of lipoproteins typically range from about 7nm to about 120 nm. The term “about” in the context of a numerical valuerepresents the value +/−10% thereof. In this diameter size range, thereexist subfractions of the particles that are important predictors ofcardiovascular disease. For example, VLDL transports triglycerides inthe blood stream; thus, high VLDL levels in the blood stream areindicative of hypertriglyceremia. These subfractions can be identifiedby analytical techniques that display the quantity of material as afunction of lipoprotein size or density.

Regarding lipoprotein density analysis, ultracentrifugally isolatedlipoproteins can be analyzed for flotation properties by analyticultracentrifugation in different salt density backgrounds, allowing forthe determination of hydrated LDL density, as shown in Lindgren, et al,Blood Lipids and Lipoproteins: Quantitation Composition and Metabolism,Ed. G. L. Nelson, Wiley, 1992, p. 181-274, which is incorporated hereinby reference. For example, the LDL class can be further divided intoseven subclasses (see Table 1) based on density or diameter by using apreparative separation technique known as equilibrium density gradientultracentrifugation. It is known that elevated levels of specific LDLsubclasses, LDL-IIIa, IIIb, IVa and IVb, correlates closely withincreased risk for CHD (i.e., coronary heart disease), includingatherosclerosis. Furthermore, determination of the total serumcholesterol level and the levels of cholesterol in the LDL and HDLfractions are routinely used as diagnostic tests for coronary heartdisease risk. Lipoprotein class and subclass distribution is a morepredictive test, however, since it is expensive and time-consuming, itis typically ordered by physicians only for a limited number ofpatients.

With respect to measurement of the sizes of lipoproteins, currentlythere is no single accepted method. Known methods for measuring thesizes of lipoproteins within a clinical setting include the verticalauto profile (VAP) (see e.g. Kulkarni, et al., J. Lip. Res., 1994,35:159-168) whereby a flow analyzer is used for the enzymatic analysisof cholesterol in lipoprotein classes separated by a short spin singlevertical ultracentrifugation, with subsequent spectrophotometry andanalysis of the resulting data. Another method (see e.g. Jeyarajah, E.J. et al., Clin Lab Med., 2006, 26:847-70) employs nuclear magneticresonance (NMR) for determining the concentrations of lipoproteinsubclasses. In this method, the NMR chemical shift spectrum of a bloodplasma or serum sample is obtained. The observed spectrum of the entireplasma sample is then matched by computer means with known weighted sumsof previously obtained NMR spectra of lipoprotein subclasses. The weightfactors that give the best fit between the sample spectrum and thecalculated spectrum are then used to estimate the concentrations ofconstituent lipoprotein subclasses in the blood sample. Another method,electrophoretic gradient gel separation (see e.g. U.S. Pat. No.5,925,229 incorporated by reference herein) is a gradient gelelectrophoresis procedure for the separation of LDL subclasses. The LDLfractions are separated by gradient gel electrophoresis, producingresults that are comparable to those obtained by ultracentrifugation.This method generates a fine resolution of LDL subclasses, and is usedprincipally by research laboratories. However, the gel separationmethod, which depends on uniform staining of all components that aresubsequently optically measured, suffers from nonuniform chromogenicity.That is, not all lipoproteins stain equally well. Accordingly, thedifferential stain uptake can produce erroneous quantitative results.Additionally, the nonuniform chromogenicity can result in erroneousqualitative results, in that measured peaks may be skewed to asufficient degree as to cause confusion of one class or subclass oflipoprotein with another. Furthermore, gradient gel electrophoresis cantake many hours to complete. It would be useful if gradient gelelectrophoresis separation times could be shortened and the analysissimplified so that high resolution lipid analysis could be used inclinical laboratories as part of a routine screening of blood samples,and for example to assign a risk factor for cardiovascular disease.

Accordingly, a high-resolution methodology for measuring all subclassesof LDL as well as VLDL, IDL (intermediate density lipoprotein), HDL,Lp(a) and chylomicron particles that is accurate, direct, and complete,would be an important innovation in lipid, including lipoprotein,measurement technology. If inexpensive and convenient, such an assaycould be employed not only in research laboratories, but also in aclinical laboratory setting. Ideally, clinicians could use thisinformation to improve current estimation of coronary disease risk andmake appropriate medical risk management decisions based on the assay.

Indeed, more recent methods for the quantitative and qualitativedetermination of lipoproteins from a biological sample have beendescribed by Benner et al. (U.S. Pub. App. No. 2003/0136680, filed Nov.12, 2002, and incorporated by reference in its entirety herein) whichmethods employ particulate size and/or ion mobility devices.

Ion mobility, also known as ion electrical mobility or charged-particlemobility, analysis offers an advantage over the other methods describedherein in that it not only measures the particle size accurately basedon physical principles but also directly counts the number of particlespresent at each size, thereby offering a direct measurement oflipoprotein size and concentration for each lipoprotein. Ion mobilityanalysis has been used routinely in analyzing particles in aerosols, andanalyzers suitable for ion mobility analysis have been adapted toanalyze large biological macromolecules. See e.g. Benner et al. (Id.)Ion mobility analysis is a very sensitive and accurate methodology with,nonetheless, a drawback that ion mobility analysis measures allparticles introduced into the system. Accordingly, it is of primeimportance to isolate and/or purify the compounds of interest prior toanalysis. Lipoproteins are candidates for this method becauselipoproteins can be isolated from other serum proteins based on densityand other features described herein. Accordingly, by the presentinvention there are provided methods for purification and isolation ofbiomolecules including, without limitation, lipoproteins and biologicalcomplexes containing lipoproteins, for use in ion mobility analysis. Thepresent invention further provides apparatus and methods for conductingion mobility analyses.

SUMMARY OF THE INVENTION

By the present invention there are provided methods for the preparationof sample for, and apparatus useful for, differential charged-particlemobility analysis (also referred to herein as “ion mobility analysis”)of lipoproteins utilizing a gas-phase electrophoretic-mobility molecularanalyzer.

In a first aspect the invention provides a method for purifyinglipoproteins suitable for ion mobility analysis of lipoprotein class andsubclass, which method includes the following steps: (a) preparing acentrifuge tube containing a first solution underneath a sample, whichsample comprises one or more lipoproteins and non-lipoproteincomponents, which first solution has a first density greater than 1.00g/mL and less than or equal to about 1.21 g/mL; and (b) subjecting thetube to centrifugation sufficient to cause the non-lipoproteincomponents to migrate toward the bottom of the tube and away from thelipoproteins, thereby providing purified lipoproteins. In someembodiments, the first density is in the range of about 1.15 g/mL toabout 1.21 g/mL.

In the context of this aspect of the invention, the sample containinglipoproteins is obtained by processing of a blood specimen from a mammalas described herein, which processing optionally includes adjustment ofdensity by the addition of salts including for example, withoutlimitation, the Cl, Br, and/or I salts of Na, K, and/or Cs.

“Centrifugation” means separation or analysis of substances in asolution as a function of density and density-related molecular weightby subjecting the solution to a centrifugal force generated byhigh-speed rotation in an appropriate instrument.

As used herein, “purify” and like terms refer to an increase in therelative concentration of a specified component with respect to othercomponents. For example without limitation, removal of lipid from alipoprotein solution constitutes purification of the lipoproteinfraction, at e.g. the expense of the lipid fraction. It is understoodthat “purifying” and like terms in the context of centrifugation refersto sufficient separation in a centrifuge tube following centrifugationto allow extraction of the separated components by methods well known inthe art including, without limitation, aspiration and/or fractionation.Surprisingly, it has been found that reducing the density oflipoprotein-containing solutions prior to centrifugation for example,without limitation, by reducing the salt concentration thereof, resultsin enhanced recovery of certain fractions of lipoprotein, including LDLand HDL fractions.

Further to this aspect of the invention are provided in certainembodiments a second solution within the centrifuge tube, above andadjacent to the sample, which second solution is preferably an aqueoussolution, more preferably water or deuterated forms thereof, of lowerdensity than the first solution. Accordingly, the density of the secondsolution is greater than or equal to 1.00 g/mL and less than the densityof the first solution. Surprisingly, it has been found that overlaying alipoprotein-containing sample in a centrifuge tube with a solutionhaving lower density results in enhanced recovery of lipoproteinfollowing centrifugal separation. Without wishing to be bound by anytheory, it is believed that ionic flow from the more denselipoprotein-containing solution to the less dense, preferably aqueous,overlaid second solution modulates the buoyancy of lipids therein,resulting in enhanced recovery of lipoprotein.

In another aspect, the invention provides yet a further method forpurifying lipoproteins, which method includes the following steps: (a)preparing a centrifuge tube containing a sample and a first solutionlocated below and adjacent the sample, the sample comprising one or morelipoproteins and non-lipoprotein components, the sample furthercomprising Reactive Green dextran and dextran sulfate (DS), the firstsolution comprising deuterium oxide (D₂O); and (b) subjecting thecentrifuge tube to centrifugation sufficient to cause thenon-lipoprotein components to migrate toward the bottom of the tube andaway from the lipoproteins. In some embodiments, the purifiedliporoteins so separated are then removed for ion mobility analysis. Insome embodiments, the density of the first solution is in the range 1.1g/mL to about 1.21 g/mL. In some embodiments, the density of the firstsolution is in the range 1.00 g/mL to about 1.10 g/mL. In someembodiments, the first solution comprises D₂O. In some embodiments, thefirst solution is substantially D₂O.

In another aspect, the invention provides methods for purifyinglipoproteins for ion mobility analysis, which methods do not includecentrifugation, which methods include the following steps: a) admixing asolution comprising lipoproteins and non-lipoproteins with one or morepolyanionic compounds and one or more divalent cations; b) allowing aprecipitate containing lipoproteins to form in the admixed solution; andc) after step b), collecting the precipitated lipoproteins andsubjecting the precipitated lipoproteins to ion mobility analysis afterresolubilization.

In another aspect, the invention provides methods for purifyinglipoproteins for ion mobility analysis, which methods do not includecentrifugation, which methods include the following steps: a) admixing asolution comprising lipoproteins and non-lipoproteins with one or morelipoprotein-capture ligands capable of binding lipoproteins to form alipoprotein/lipoprotein-capture ligand complex; b) isolating thelipoprotein/lipoprotein-capture ligand complex; and c) releasing thelipoproteins from the lipoprotein/lipoprotein-capture ligand complex andsubjecting the lipoproteins to ion mobility. In some embodiments, thelipoproteins are selected from the group consisting of HDL, LDL, Lp(a),IDL and VLDL. In some embodiments, the lipoprotein-capture ligand isselected from the group consisting of aptamer and antibody. In someembodiments, the lipoprotein-capture ligand is an antibody.

Further any of the aspects contemplating isolation and/or purifying oflipoproteins described herein, in another aspect the invention providesmethods for analyzing the size distribution of lipoproteins, whichmethod includes the following steps: (a) providing one or morelipoproteins in accordance with any of the methods described herein; and(b) subjecting the one or more lipoproteins to ion mobility analysis,thereby determining the size distribution of the lipoproteins. In afurther embodiment, this method is used to determine in a patient samplethe concentration of HDL, LDL, IDL, and VLDL and more preferably HDL,LDL, IDL, VLDL and Lp(a). The patient sample is preferably plasma orserum. The method may also include use of an internal standard such asone or more labeled lipoproteins (e.g. fluorescent label) to monitorsample loss during processing so as to obtain more accuratedeterminations of lipoprotein concentration in the starting sample to beevaluated.

In another aspect of the invention, an apparatus for differentialmobility analysis comprises one or more pumps adapted to transportsample through a capillary, an ionizer adapted to charge particles ofthe sample as the sample flows within the capillary, and an ion mobilityanalyzer adapted to perform a differential mobility analysis on thesample of charged particles. The ionizer may include a conductive unionaround a part of the capillary. In one embodiment, the conductive unionforms a microtite region in a part of the capillary and applies a chargeto the sample flowing therethrough, thereby charging particles of thesample.

Certain embodiments of the apparatus further comprise an autosampleradapted to provide a sample for differential mobility analysis to theone or more pumps.

In some embodiments, the one or more pumps include a high-flow pumpadapted to provide the sample to a nanoflow pump, the nanoflow pumpbeing adapted to provide the sample to the capillary. The high-flow pumpmay pump sample at a rate of approximately 15-25 microliters per minute,and the nanoflow pump may pump the sample at a rate of approximately100-200 nanoliters per minute.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of density on lipoprotein recovery from a plasmasample during a 3.7 hr ultracentrifugation. Samples were prepared induplicate using different density solutions and centrifugation for 3.7hr. After collecting the lipoprotein fraction, it was dialyzed beforeanalysis by Ion Mobility. Each panel shows the profile of eachreplicate. Solution densities: A=1.23 g/mL; B=1.181 g/mL; C=1.170 g/mL;D=1.165 g/mL. The abscissa is lipoprotein diameter (nm), and theordinate is an arbitrarily scaled mass coordinate, which mass coordinateis linearly related to the actual number of particles counted as afunction of size (i.e., diameter).

FIG. 2 shows a comparison of lipoprotein recovery from plasma usingeither a D₂O or a low salt protocol (without D₂O) in a centrifugationseparation experiment. Dark profile reflects a 2 hr centrifugation usingD₂O as the dense solution (1.107 g/mL). Light profile reflects a 3.7 hrcentrifugation using KBr as the dense solution (1.151 g/mL). A—indicatespeak height of albumin for 2 hr centrifugation; B— indicates the albuminpeak height for 3.7 hr centrifugation. The abscissa is lipoproteindiameter (nm), and the ordinate is an arbitrarily scaled masscoordinate, as discussed in the legend for FIG. 1.

FIG. 3 shows the result of Apo A1, Apo B and total cholesterol (TC)recovery from plasma using D₂O in combination with RGD/DS [RGD: ReactiveGreen 19 (RG 19) conjugated with dextran; RGD/DS: RGD in combinationwith DS] in a centrifugation separation experiment. Abscissa indicatesanalyte measured. Numbers associated with each box refer to a uniquepatient identification numbering system.

FIG. 4 shows the result of lipoprotein recovery from plasma aftercentrifugal purification using RGD. RGD was added to samples at variousconcentrations and centrifuged for 2 hr 15 min using D₂O as the densesolution. Albumin Peak heights are indicated for the four differentconcentrations of RGD used; A, 10 mg/mL RGD; B, 15 mg/mL RGD; C, 20mg/mL RGD; and D, 25 mg/mL RGD. The abscissa is lipoprotein diameter(nm), and the ordinate is an arbitrarily scaled mass coordinate, asdiscussed in the legend for FIG. 1.

FIG. 5 shows the result of lipoprotein recovery from plasma aftercentrifugal purification with RGD and ethylenediaminetetracidic acid(EDTA), or with RGD/DS and EDTA, optionally ammonium acetate (AA).Legend: (B) extraction with 7.5 mg/mL RGD and 2.5 mg/mL DS, dilutionwith 25 mM ammonium acetate; (A) extraction with 7.5 mg/mL RGD and 2.5mg/mL DS, dilution with 25 mM ammonium acetate with 5 ug/mL DS; (D)extraction with 7.5 mg/mL RGD, dilution with 25 mM ammonium acetate; (C)extraction with 7.5 mg/mL RGD, dilution with 25 mM ammonium acetate with5 ug/mL DS. The abscissa is lipoprotein diameter (nm), and the ordinateis an arbitrarily scaled mass coordinate, as discussed in the legend forFIG. 1.

FIG. 6 shows the result of inclusion of DS in the dilution bufferfollowing traditional density separation and dialysis. A and B: 5 ug/mLDS included in the ammonium acetate dilution buffer. C and D: no DS inthe ammonium acetate dilution buffer. The abscissa is lipoproteindiameter (nm), and the ordinate is an arbitrarily scaled masscoordinate, as discussed in the legend for FIG. 1.

FIG. 7 shows the resulting lipoprotein profile in conjunction with atypical report on lipoprotein fractionation by ion mobility. Theabscissa is lipoprotein diameter (nm), and the ordinate is a mass,calculated from ion mobility data and parameters as known in the art.Areas shown with cross-hatching indicate relative risk, with thediagonal-lined sections representing medium risk, vertical-linedsections representing lower risk, cross-hatched sections representinghigher risk, and the shaded sections representing indeterminate risk.

FIG. 8 illustrates an apparatus for ion mobility analysis according toan embodiment of the present invention.

FIGS. 9A and 9B illustrate embodiments of conjunctive unions for usewith the apparatus of FIG. 8.

DETAILED DESCRIPTION OF THE INVENTION

“VLDL, IDL, LDL, and HDL” refer to classifications of lipoproteins asshown in Table 1. It is understood that the values used in Table 1 forsizes are determined by gel electrophoresis methods, as known in theart. With the ion mobility analysis methods disclosed here, it has beenobserved that all measurements of lipoprotein diameter obtained with ionmobility analysis are shifted to smaller diameters compared to the dataobtained with gel electrophoresis. Without wishing to be bound by anytheory, it is believed that this difference is due to calibration of thegels. The shift appears to be linearly related and approximated by thefollowing formula:0.86*gel diameter=IM diameter

Table 1 describes the standard classes and subclass designationsassigned to various lipoprotein fractions using traditional gelelectrophoresis measurements: very low density lipoproteins (VLDLs) withsubclasses VLDL I and II; intermediate density lipoproteins (IDLs) withsubclasses IDL I and II; low density lipoproteins (LDLs) with subclassesI, IIa, IIb, IIIa, IIIb, IVa and IVb; and high density lipoproteins(HDLs), which typically includes several subclasses, such as HDL IIa,IIb, IIIa, IIIb, and IIIc.

TABLE 1 Major Lipoprotein Class, Subclass, Density and Particle SizeClass Acronym Name Density Particle Diameter Subclass (g/mL) (Å) VLDLVery Low Density Lipoprotein I <1.006 330-370 II 1.006-1.010 300-330 IDLIntermediate Density Lipoprotein I 1.006-1.022 285-300 II 1.013-1.019272-285 LDL Low Density Lipoprotein I 1.019-1.023 272-285 IIa1.023-1.028 265-272 IIb 1.028-1.034 256-265 IIIa 1.034-1.041 247-256IIIb 1.041-1.044 242-247 IVa 1.044-1.051 233-242 IVb 1.051-1.063 220-233HDL High Density Lipoprotein IIa 1.063-1.100  98-130 IIb 1.100-1.12588-98 IIIa 1.125-1.147 82-88 IIIb 1.147-1.154 77-82 IIIc 1.154-1.20372-77

Without wishing to be bound by any theory, it is believed that theobserved differences between ion mobility analysis diameters and gelelectrophoresis diameters may also be due to distortion of lipoproteinsinteracting with the gel matrix under the influence of the intrinsicimpressed electric field of the electrophoresis gel. The size differencemay also be due to historical data used to convert particle density(obtained from analytic ultracentrifuge separations) to particle sizeobtained from electron microscopy.

“Chylomicrons” means biological particles of size 70-120 nm, withcorresponding densities of less than 1.006 g/mL. Chylomicrons have notbeen found to have any clinical significance in the prediction of heartdisease, for example CHD.

“Apo A” as known in the art is a protein component of HDL. “Apo B” is aprotein component of LDL, IDL, Lp(a), and VLDL, and indeed is theprimary apolipoprotein of lower density lipoproteins, having humangenetic locus 2p24-p23, as known in the art.

“Albumin” refers to ubiquitous proteins constituting approximately 60%of plasma, having density about 1.35 g/mL, as known in the art.

“Lp(a),” and “lipoprotein (a)” refer to a type of lipoprotein found inserum having a molecular composition distinct from IDL and LDL, which isfound in complex with apolipoprotein a [apo(a)]. Lp(a) has a particlesize that overlaps with LDL and IDL and therefore can interfere withparticle size analysis when Lp(a) particles are present in the sample.Although some patients have naturally occurring low Lp(a)concentrations, it is believed to be good practice to remove the Lp(a)prior to LDL size measurements to preclude otherwise inaccuratemeasurements for those patients having significant Lp(a) concentrations.In this manner, potential Lp(a) size interference problems can beavoided.

The present invention contemplates methods of ion mobility, andpreparation of samples for ion mobility analysis. Ion mobility utilizesthe principle that particles of a given size and charge state behave ina predictable manner when carried in a laminar-air flow passed throughan electric field. Accordingly, ion mobility analysis is a technique todetermine the size of a charged particle undergoing analysis when thecharged particle is exposed to an electric field.

Electrical mobility is a physical property of an ion and is related tothe velocity an ion acquires when it is subjected to an electricalfield. Electrical mobility, Z, is defined as

$\begin{matrix}{Z = \frac{V}{E}} & (1)\end{matrix}$where V=terminal velocity and E=electrical field causing particlemotion. Particle diameter can be obtained from

$\begin{matrix}{Z = \frac{{neC}_{c}}{3{\pi\eta}\; d}} & (2)\end{matrix}$where n=number of charges on the particle (in this case a singlecharge), e=1.6×10⁻¹⁹ coulombs/charge, C_(c)=particle size dependent slipcorrection factor, .η=gas viscosity, and d=particle diameter.Accordingly, solving for d, provides the following relationship:

$\begin{matrix}{d = {\frac{{neC}_{c}}{3{\pi\eta}}{\frac{E}{V}.}}} & (3)\end{matrix}$

Thus, an explicit relationship for particle diameter as a function ofknown parameters results. By setting the parameters to different values,different particle diameters of the charged particles may be selected asfurther described below and known in the art. In preferred methods ofion mobility analysis, the electric field strength E acting upon thecharged particle is varied during analysis.

In ion mobility analysis, particles (e.g., lipoproteins and the like)are carried through the system using a series of laminar airflows. Thelipoproteins in a volatile solution are introduced to an electrospraychamber containing approximately 5% CO₂ wherein the lipoproteinsdesolvate. In the electrospray chamber the desolvated, chargedlipoproteins are neutralized by ionized air, introduced for examplewithout limitation by an alpha particle emitter in the chamber. Based onFuch's formula, a predictable proportion of particles emerge from thechamber carrying a single charge and are transported from the chamber tothe Differential Mobility Analyzer (DMA). For details on Fuch's formula,reference is made to Fuchs, N. A.: The Mechanics of Aerosols, Macmillan,1964. “Differential Mobility Analyzer,” “DMA” and like terms refer todevices for classifying charged particles on the basis of ion electricalmobility, as known in the art and described herein. In ion mobilityanalysis, when particles have a known uniform charge, the size of theparticles classified may be determined from the mobility thereof. In theDMA the particles enter at the top outer surface of the chamber and arecarried in a fast flowing laminar-air flow, (i.e., “the sheath flow”).The sheath flow is filtered (to remove particles) air that constantlyrecirculates through the DMA at a constant velocity of 20 L/min. As theparticles pass through the DMA (carried in the sheath flow) the electricpotential across the chamber is ramped up at a known rate. As theelectrical potential changes, particles of different diameter arecollected via a slit at the bottom inner surface of the chamber.Particles follow a non-linear path through the DMA depending on theircharge and diameter. At any given electrical potential, particles ofknown size will follow a path that will allow them to pass through thecollecting slit. Particles passing through the collecting slit arepicked up by another, separate laminar-flow air stream and are carriedto a particle counter. The particle counter enlarges the particles bycondensation to a size that can be detected and counted for example by alaser detection system. Knowing the electrical potential being appliedto the DMA when the particle was collected permits the accuratedetermination of the particle diameter and the number of particlespresent at that size. This data is collected and stored in bins as afunction of time for different particle size. In this way the number ofparticles of any given size range can be determined and converted to aconcentration of particles based on the time required to collect thedata, the flow rate of sample being introduced into the electrospraydevice, and the number of charged particles at that size.

In methods of the present invention contemplating isolation and/orpurification of lipoproteins, initial sample collection and preparationmay be conducted by methods well known in the art. Typically, a 2 to 5ml fasting blood specimen is initially taken. Chylomicrons are nottypically present in subjects who have been fasting for a period of atleast 12 hours; thus, overlap of VLDL sizes and chylomicron sizes iseliminated by fasting. The specimen is then initially spun in acentrifuge (e.g., clinical centrifuge) preferably for approximately 10minutes at approximately 2000×G, which centrifugation is sufficient toremove the cellular components from the specimen. During this process,the more dense cellular components stratify at the bottom of the sample.A remaining less dense plasma specimen containing lipoproteins on top isthen drawn off using methods well known in the art, e.g., aspiration.

Historically, in preparation for centrifugation, a plasma specimen couldbe density-adjusted to a specific density using high purity solutions orsolids of inorganic salts, e.g., sodium chloride (NaCl), sodium bromide(NaBr) and the like. In some previous protocols, the specific densitywould be chosen to be greater than or equal to the highest density ofthe lipoprotein material to be analyzed, so that the lipoproteinmaterial would float when density stratified. “Density stratified” andlike terms refer to the layering of components in a solution subjectedto centrifugation. These densities are tabulated in Table 1, table oflipoprotein classes, subclasses, densities, and sizes. Thedensity-adjusted sample could then be ultracentrifuged for example forapproximately 18 hours at 100,000×G to separate the non-lipoproteinproteins from the lipoproteins. Non-lipoprotein proteins, particularlyalbumin, are removed from the plasma specimen, preferably by thisultracentrifugation step. The lipoproteins float to the top of thesample during ultracentrifugation. Accordingly, by sequentiallycentrifuging from lowest density to highest density of the densityadjustment, the various classes and subclasses of lipoproteins could besequentially extracted. Typically, a dialysis step would be requiredfollowing extraction of a centrifuged sample to remove salts introducedfor adjustment of density, which dialysis step would typically require4-12 hrs under conditions well known in the art.

Conditions for centrifugation for lipoprotein-containing samplesdescribed herein are well known in the art of biochemical separation.For example without limitation, samples are typically centrifuged at 10C for 1-4 hrs at 223,000×G. In some embodiments, centrifugation employscentrifugal force of 50,000-100,000, 100,000-120,000, 120,000-150,000,150,000-200,000, 200,000-230,000, 230,000-250,000×G, or even higherforce. In some embodiments, the time of centrifugation is 1, 2, 2.2,2.4, 2.6, 2.8, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 hr, or even longer. Priorto analysis by ion mobility, an aliquot of the lipid fraction is removed(e.g., 10-200 μL) from the top of the centrifuge tube and diluted (e.g.,1:800) in 25 mM ammonium acetate (AA), 0.5 mM ammonium hydroxide, pH7.4. Advantageously, in some embodiments described herein, a dialysisstep is not necessary in conjunction with methods of the invention,resulting in less time required for analysis.

In embodiments of the invention which contemplate lipoproteins, thelipoproteins are selected from the group consisting of HDL, LDL, IDL,Lp(a), and VLDL. In some embodiments, the lipoproteins are HDL.

In some embodiments of aspects provided herein which contemplatelipoproteins, the lipoproteins may derive from a plasma specimen,obtained by methods well known in the art or as described herein. Theterms “biological specimen,” “biological sample” and like terms refer toexplanted, withdrawn or otherwise collected biological tissue or fluidincluding, for example without limitation, whole blood, serum andplasma. The term “plasma” in the context of blood refers to the fluidobtained upon separating whole blood into solid and liquid components.The term “serum” in the context of blood refers to the fluid obtainedupon separating whole blood into solid and liquid components after ithas been allowed to clot. In some embodiments of any of the aspects ofthe present invention, the biological specimen is of human origin. Insome embodiments of any of aspects provided herein, the biologicalspecimen is serum. In some embodiments of any of the aspects providedherein, the biological specimen is plasma.

In some embodiments of the invention which contemplate centrifugation,the centrifugation does not reach equilibrium. “Centrifugationequilibrium” and like terms refers to centrifugation conducted forsufficient time and at sufficient centrifugal force such that thecomponents of the solution being centrifuged have reached neutraldensity buoyancy, as well known in the art. Surprisingly, it has beenfound that foreshortened centrifugation protocols, as described hereinwherein centrifugal equilibrium is not reached, can nonetheless providesignificant purification of lipoproteins.

In some embodiments of the invention which contemplate centrifugation ofsample containing lipoproteins and non-lipoprotein components, purifiedlipoprotein is collected from the top portion of the centrifuge tubefollowing centrifugation. “Top portion of the centrifuge tube” and liketerms refer to the liquid in the upper portion of a centrifuge tube whenviewed outside of the centrifuge rotor which may, but does notnecessarily, include liquid at the very top.

Further any of the methods of the present invention directed topurifying lipoproteins, it has been surprisingly found that reduction ofthe density of the solution to a value less or equal to about 1.21 g/mLwhile centrifuging to less than equilibrium actually results in improvedrecovery, hence purification, of LDL and HDL.

Exemplary ion mobility results for lipoproteins from plasma samplespurified by centrifugation with varying densities of solution are shownin FIG. 1. In these experiments, serum samples (25 uL) were overlaid ona cushion (200 uL) of four different density salt (KBr) solutions. Thedensities of the solutions were 1.165, 1.170, 1.181, and 1.23 g/mL. Eachsample was ultracentrifuged for a period of 3.7 hr at 223,000×G. The top100 uL after the centrifugation was removed. Fractionated lipoproteinsamples from each density were dialyzed overnight against ammoniumacetate (25 mM), ammonium hydroxide (0.5 mM), pH 7.4. Following dialysiseach sample was analyzed by ion mobility with the resulting profilesshown in FIG. 1. A reduction is apparent in the lipoprotein profiles inthe HDL region seen at the lower densities compared to 1.23 g/mL.Without wishing to be bound by any theory, this observation is believeddue to more efficient removal of plasma proteins with lower saltsolutions.

With further reference to FIG. 1, the abscissa is the particle size(i.e., diameter), and the ordinate is an arbitrarily scaled mass. Thearea under the curves, in a particle mass versus independent variable(such as size, density, mobility, etc.) distribution, is directlyrepresentative of the lipoprotein particle mass. The measurementtechnique relies on counting individual particles as a function of size(diameter). It is therefore possible to convert the number of particlesat a specific size into a mass value using the volume and density of theparticles. The density of lipoproteins is a well-known function ofparticle size and is obtainable for example from the literature. Themass values associated with the figure are simply scaled to indicaterelative values but can be converted to actual mass of lipoproteins inplasma using dilution factors along with flow rates of sample and airpassing through the ion mobility spectrometer. Accordingly, in someembodiments adjusting the density of a lipoprotein-containing solutionprior to non-equilibrium centrifugation to a value lower than expectedto separate the higher density lipoproteins (e.g., HDL) actually resultsin separation of HDL and LDL. Advantageously, the method of reducing thedensity of the lipoprotein-containing sample also results in increasedseparation from albumin.

In some embodiments of aspects provided herein contemplatingcentrifugation of a sample containing lipoproteins and non-lipoproteincomponents, the first solution comprises D₂O. In some embodiments, thedensity of the first solution is determined substantially by the contentof D₂O, wherein the first solution has a density in the range 1.00 toabout 1.10 g/mL. The density of D₂O is approximately 1.107 gm/mL at 25C. Accordingly, in some embodiments of the invention, the aqueouscomponent includes 0-99% D₂O, or even higher. In some embodiments, theamount of D₂O is in the range, for example without limitation, 10-99,20-99, 30-99, 40-99, 50-99, 10-90, 20-90, 30-90, 40-90, 50-90%, and thelike. In some embodiments, the content of D₂O is a specific value, forexample without limitation, about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 96, 97, 98, 99, or even 100% D₂O. In some embodiments, thefirst solution is substantially D₂O. The term “essentially D₂O” refersto D₂O comprising the aqueous component with no additionally added H₂O.The terms “substantially D₂O” and like terms refer to D₂O content in arange greater than 50%, for example without limitation, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or even 100% D₂O.

In some embodiments of this aspect, the lipoprotein-containing sampleincludes little if any added salt. With reference to FIG. 2(experimental conditions provided in Example 1), which shows the resultof a centrifugation procedure conducted using D₂O and no additionalsalts for density adjustment, and a low-density salt solution withoutD₂O, approximately equivalent recovery of LDL and certain HDL fractions(e.g., HDL-IIb and HDL-IIa) was observed after 2 hrs (D₂O) and 3.7 hrs(low-density salt solution). With further reference to FIG. 2, theprofiles for D₂O and low-density salt centrifugation procedures resultin similar profiles, with nonetheless increased albumin (peak at startof HDL 3 region) judged due to decreased centrifugation time with theD₂O sample. Without wishing to be bound by any theory, it appears thatreduction of salt content with concomitant increase in density using D₂Oresults in shorter time required for centrifugation and purification oflipoprotein from a lipoprotein-containing sample.

With reference to FIG. 3, under the conditions employed for FIG. 3(experimental conditions of Example 2) approximately equal recovery ofApo A1 and Apo B after centrifugation are observed, indicating thatlower density obtained with D₂O does not result in selective recovery oflarger less dense particles.

In some embodiments of the method of the present invention directed topurifying lipoproteins by the placement of a less dense solution aboveand adjacent to a lipoprotein-containing sample solution prior tocentrifugation, a single density adjustment of thelipoprotein-containing solution is conducted using inorganic salts,preferably NaCl and/or NaBr. For example, over this sample in acentrifuge tube can be layered a second solution having density lessthan the density of the lipoprotein-containing sample solution.Alternatively, the lipoprotein-containing sample solution can beintroduced under the second solution in the centrifuge tube. Thelipoprotein-containing sample density adjustment can be selected withinthe range of 1.00 to about 1.21 g/mL according to the densities in Table1 to separate a class of lipoproteins having equal or lesser density.The density of the second solution can be selected within the range 1.00g/mL up to just less than the density of the lipoprotein-containingsample solution, preferably in the range 1.00 to about 1.15 g/mL, morepreferably 1.00 g/mL. In this manner, the HDL, IDL, LDL, Lp(a) and VLDLlipoproteins having densities less than the density of thelipoprotein-containing sample solution can be simultaneously extracted.Surprisingly, it has been found that providing a lipoprotein-containingsolution in a centrifuge tube with a solution having lower density aboveand adjacent the lipoprotein-containing solution results in enhancedrecovery of lipoprotein employing centrifugal separation. In preferredembodiments, lipoprotein containing fractions are withdrawn from thevery top of the tube, at the meniscus, downward to the appropriatedesired volume.

Further to these methods, it has been found that the time required forcentrifugal separation of a lipoprotein-containing sample is reduced inlower density samples when compared to a corresponding period of timerequired for higher density samples. The terms “corresponding period oftime” and the like in the context of centrifugal separation refer to thelength of time of centrifugation required to achieve a specified levelof separation, given equivalent centrifugal force during thecentrifugation. For example without limitation, it has been found thatat least 2 hrs centrifugation (at e.g., 230,000×G) is required to removesufficient albumin from a typical lipoprotein-containing sample, withless centrifugation time resulting in less removal of albumin. Withoutwishing to be bound by any theory, it appears that by lowering thedensity of the sample, albumin, and indeed other non-lipoprotein plasmaproteins, are more readily stratified and thus separated fromlipoprotein. Accordingly, a key factor in optimizing purification oflipoprotein is shortening the time of centrifugation to maximize theloss of albumin and other plasma proteins while retaining HDL.

In some embodiments of aspects provided herein which contemplatecentrifugation of sample containing lipoproteins and non-lipoproteincomponents, the sample further comprises a compound which can act as aprecipitant for selected lipoprotein components therein, as known in theart. “Precipitant” refers to a compound which may cause or promoteprecipitation of a biomolecule upon addition to a solution of suchbiomolecule. A precipitant may require an additional agent to affordprecipitation. “Additional agent to afford precipitation” and like termsrefer to compounds which act with a precipitant and may be required toafford precipitation by the precipitant. Exemplary precipitants include,without limitation, salts of charged inorganic ions, preferably ammoniumsulfate, antibodies, charged polymers (e.g., DS and the like) optionallyin the presence of ionic species (e.g., divalent cations), lectins, andthe like. In some embodiments, the precipitant is present albeit underconditions (e.g., pH, concentration, lack of necessary additionalagents, and the like) wherein lipoproteins are not precipitated. In someembodiments, the precipitant is DS. In some embodiments, the precipitantis DS, and the necessary additional agent is a divalent cation. In someembodiments, the lipoprotein-containing sample comprises DS but lacksdivalent cations. Without wishing to be bound by any theory, it isbelieved that DS binds to particles which contain lipids in the presenceof divalent cations, and that DS binding may interfere with non-specificbinding interactions with resulting enhancement of recovery of certainlipoproteins. For example without limitation, it is observed thatinclusion of DS significantly improved recovery of LDL from somepreparations described herein.

In some embodiments of aspects provided herein which contemplatecentrifugation of sample containing lipoproteins and non-lipoproteincomponents, the sample further comprises an albumin-binding compoundunder conditions suitable to allow formation of a complex comprisingalbumin and albumin-binding compound. Representative albumin-bindingcompounds include, without limitation, aromatic albumin-binding dyes.The aromatic albumin-binding dye may comprise a diazo dye; an alkalimetal salt, alkaline earth metal salt, or amine salt of said diazo dye;a sulfonic acid dye; a physiologically-acceptable alkali metal salt,alkaline earth metal salt, or amine salt of said sulfonic acid dye; ormixtures thereof. Aromatic albumin-binding dyes particularly useful inthe present invention include Reactive Blue 2, Evans Blue, Trypan Blue,Bromcresol Green, Bromcresol Purple, Methyl Orange, Procion red HE 3B,and the like. In certain embodiments, the albumin-binding compound is ananalog of nicotinamide adenine dinucleotide (NAD). Representative NADanalogs suitable for use as albumin-binding compounds include, withoutlimitation, RG 19, and Cibacrom Blue 3GA (CB 3GA).

In embodiments of the method contemplating the use of albumin-bindingcompounds, after mixture of the albumin-binding compound with alipoprotein-containing sample, the sample is centrifuged as describedherein. In some embodiments, the albumin-binding compound is conjugatedwith a chromatographic medium, which conjugate promotes facile removalof albumin complexed with albumin-binding compound for example, withoutlimitation, by filtration. In some embodiments, the conjugatedalbumin-binding compound is observed to stratify at the bottom of thecentrifuge tube, thereby facilitating removal (e.g., by aspiration,etc.) of the lipoprotein-containing fraction. In some embodimentswherein the albumin-binding compound is conjugated with achromatographic medium, the chromatographic medium may be paramagneticparticles, dextran, agarose or Sephadex®, preferably dextran“Paramagnetic particle” as known in the art refers to particles having amagnetite core coated with a ligand, for example without limitation,streptavidin. The affinity of biotin for streptavidin (K_(d)=10⁻¹⁵ M) isone of the strongest and most stable interactions in biology. Thus,paramagnetic particles combine convenient magnetic separation technologywith the versatility and high affinity of the interactions such as thebiotin-streptavidin interaction. It is observed that dextran conjugatedalbumin-binding compounds tend to remain soluble longer than otherconjugate chromatographic media described herein. Without wishing to bebound by any theory, it is believed that the longer an albumin-bindingcompound can interact with albumin in a lipoprotein-containing sample,the more albumin-containing complex will be formed, thereby increasingpurity and recovery of lipoprotein.

In further embodiments of the method contemplating the use ofalbumin-binding compounds, the albumin-binding compound is presentduring centrifugation at a concentration of up to 50 mg/mL, or evenhigher, without significant change in the quantity and relativeproportion of the lipoproteins recovered from a plasma sample. Forexample, referring to FIG. 4, ion mobility analyses of alipoprotein-containing sample in which varying amounts of RG 19 wereincluded prior to centrifugation show that inclusion of RG 19 conjugatedwith dextran (RGD) results in recovery of lipoprotein with little, ifany, effect on the distribution of lipoproteins; compare FIG. 1 withFIG. 4. With reference to Example 3 and FIG. 4, while the ion mobilityprofiles of HDL and LDL are similar, there is a decrease in the size ofthe peak (albumin) at the onset of the HDL 3 peak with increasing RGDconcentration. Furthermore, the height of the peak at the higherconcentrations is similar to that seen in preparations from lowerdensity salt and 3.7 hr spins. In other embodiments, the concentrationof albumin-binding compound is for example, without limitation, 1, 2, 5,10, 15, 20, 25, 30, 35, 40, 45 or even 50 mg/mL.

In certain embodiments, the invention provides for the use of analbumin-binding compound in combination with DS. Referring to FIG. 5,use of RGD, optionally DS, optionally ammonium acetate (AA), resulted inmodulation of the recovery of LDL and HDL fractions as judged by ionmobility analysis. With reference to Example 4 and FIG. 5, there aresimilarities in the HDL region of the profiles shown in FIG. 5 withincreased recovery of HDL when DS is present in the extraction (see“7.5/2.5” vs “7.5” legend entries in FIG. 5). Additionally, low albuminpeak height is observed. It is believed that the increased peak in onepreparation in HDL 2a (FIG. 5) is not typical of the reproducibility.Also of significance is the increased recovery of LDL. Without wishingto be bound by any theory, results herein suggest that DS present in theextraction and diluent affords the best recovery and reproducibility.

In certain embodiments, purified lipoprotein-containing sample obtainedby methods of the invention are further diluted prior to ion mobilityanalysis. Referring to FIG. 6 and Example 5, the effect of presence orabsence of DS (+/−5 ug/mL) in a 1:200 dilution step with 25 mM ammoniumacetate prior to ion mobility analysis was assessed. As shown in FIG. 6,there is a significant increase in the LDL peak height in the presenceof DS, whereas HDL peak profile are relatively unaffected.

In certain aspects and embodiments, the invention contemplates methodsemploying an albumin-binding compound conjugated with chromatographicmedia in combination with DS, and further in combination with a D₂Osolution under and adjacent a lipoprotein-containing sample in acentrifuge tube. A typical procedure employing this protocol is providedin Example 6.

In some embodiments of aspects provided herein which contemplatecentrifugation of sample containing lipoproteins and non-lipoproteincomponents, the sample further comprises a non-lipoprotein captureligand capable of binding non-lipoprotein component to form anon-lipoprotein/non-lipoprotein capture ligand complex, further whereinthe centrifugation causes the non-lipoprotein/non-lipoprotein captureligand complex to be separated from the lipoprotein components.“Non-lipoprotein capture ligand” and like terms refer to compounds whichbind plasma components which are not lipoproteins. Exemplarynon-lipoprotein capture ligands include, without limitation, antibodiesand aptamers as understood in the art. For example without limitation,separation of antibody (i.e., as non-lipoprotein capture ligand) fromantigen (i.e., non-lipoprotein) can be realized with a variety ofmethods including modulation of temperature, pH, salt concentration andthe like. For further example without limitation, separation of aptamer(i.e., as non-lipoprotein capture ligand) from aptamer target (i.e.,non-lipoprotein) can be realized with a variety of methods includingmodulation of temperature, pH, salt concentration, DNase or RNase andthe like.

In some embodiments of aspects provided herein which contemplatecentrifugation of sample containing lipoproteins and non-lipoproteincomponents, the sample further comprises a lipoprotein-capture ligandcapable of binding lipoprotein component to form alipoprotein/lipoprotein-capture ligand complex, further wherein thecentrifugation causes the lipoprotein/lipoprotein-capture ligand complexto be separated from the non-lipoprotein components.“Lipoprotein-capture ligand” and like terms refer to compounds whichbind lipoproteins. Exemplary lipoprotein-capture ligands include,without limitation, antibodies and aptamers as understood in the art. Inpreferred embodiments, the lipoprotein-capture ligand is an antibody.

In some embodiments of aspects provided herein which do not contemplatecentrifugation of sample containing lipoproteins and non-lipoproteincomponents, the method contemplates a lipoprotein-capture ligand capableof binding lipoprotein component to form alipoprotein/lipoprotein-capture ligand complex.

The term “aptamer” refers to macromolecules composed of nucleic acid,such as RNA or DNA, that bind tightly to a specific molecular target.The terms “bind,” “binding” and the like refer to an interaction orcomplexation resulting in a complex sufficiently stable so as to permitseparation. In some embodiments, the aptamer specifically bind Apo A1,Apo B, or Apo(a). Methods for the production and screening of aptamersuseful for the present invention are well known in the art; see e.g.,Griffin et al., U.S. Pat. No. 5,756,291, incorporated herein byreference in its entirety and for all purposes.

As practiced in the art, the method of selection (i.e., training) ofaptamer requires a pool of single stranded random DNA oligomerscomprising both random sequences and flanking regions of known sequenceto serve as primer binding sites for subsequent polymerase chainreaction (PCR) amplification. Such DNA oligomers are generated usingconventional synthetic methods well known in the art. As an initial andoptional step, PCR amplification is conducted by conventional methods,and the amplified pool is left as duplex DNA, or used as single strandedDNA after strand separation. Optionally, transcription into RNA can beconducted. The term “oligomer pool” in this context refers to suchsingle stranded or duplex DNA, or RNA transcribed therefrom. The term“refined oligomer pool” refers to an oligomer pool which has beensubjected to at least one round of selection as described herein.

Further the aforementioned aptamer training, a “selection” step isconducted employing a column or other support matrix (i.e.,target-coupled support) having target molecule attached thereon.Attachment, well known in the art, may be by covalent or noncovalentmeans. The oligomer pool, or refined oligomer pool, and target-coupledsupport are incubated in order to permit formation ofoligonucleotide-target complex, and the uncomplexed fraction of theoligomer pool or refined oligomer pool is removed from the supportenvironment by, for example, washing by methods well known in the art.Subsequent removal of oligonucleotide by methods well known in the artresults in a refined oligomer pool fraction having enhanced specificityfor target relative to a predecessor oligomer pool or refined oligomerpool.

Alternatively, the aforementioned aptamer training can employ a “reverseselection” step wherein aptamer is selected to bind to otherconstituents of the biological sample. In this case, a column or othersupport matrix is employed (i.e., constituent-coupled support) havingother constituents of the biological sample attached thereon. Theoligomer pool, or refined oligomer pool, and constituent-coupled supportare incubated in order to permit formation ofoligonucleotide-constituent complex, and the uncomplexed fraction of theoligomer pool or refined oligomer pool is removed from the supportenvironment by, for example, washing by methods well known in the art.Subsequent removal of oligonucleotide by methods well known in the artresults in a refined oligomer pool fraction having enhanced specificityfor other constituents of the biological sample relative to apredecessor oligomer pool or refined oligomer pool. Examples of otherconstituents of the biological sample used in the reverse selection stepinclude, without limitation, immunoglobulins and albumins.

In a typical production training scheme, oligonucleotide recovered aftercomplexation with target or other constituent of the biological sampleis subjected to PCR amplification. The selection/amplification steps arethen repeated, typically three to six times, in order to provide refinedoligomer pools with enhanced binding and specificity to target or otherconstituent of the biological sample. Amplified sequences so obtainedcan be cloned and sequenced. Optionally, when a plurality of individualaptamer sequence specific for a target having been obtained andsequenced, pairwise and multiple alignment examination, well known inthe art, can result in the elucidation of “consensus sequences” whereina nucleotide sequence or region of optionally contiguous nucleotides areidentified, the presence of which correlates with aptamer binding totarget. When a consensus sequence is identified, oligonucleotides thatcontain the consensus sequence may be made by conventional synthetic orrecombinant means.

The term “antibody” refers to an immunoglobulin which binds antigen(e.g., lipoprotein or other component of the sample) with high affinityand high specificity. In this context “high affinity” refers to adissociation constant of, for example without limitation, 1 μM, 100 nM,10 nM, 1 nM, 100 pM, or even more affine, characterizing the bindingreaction of antibody with antigen to which the antibody has been raised.The term “raised” refers to the production of high affinity antibody bymethods long known in the art. Further in this context, the term “highspecificity” refers to a preference of binding of a target antigen by atest antibody relative to non-target antigen characterized by a ratio ofdissociation constants of, for example without limitation, 1, 2, 5, 10,20, 50, 100, 200, 500, 1000, 10000, or more, in favor of binding of thetarget antigen to which the test antibody has been raised.

Methods of derivatization of antibodies and aptamers contemplated by thepresent invention include, for example without limitation,biotinylation. In some embodiments, the antibody or aptamer isbiotinylated such that subsequent isolation on an avidin conjugatedmatrix, for example without limitation, an avidin chromatography column,affords facile separation by methods well known in the art ofbiochemical purification. In some embodiments, the biotinylated antibodyor aptamer in complex with a lipoprotein is further subjected tostreptavidin-conjugated magnetic beads. The ternarylipoprotein-biotinylated affinity reagent-streptavidin conjugatedmagnetic bead complex is then isolated by immunomagnetic methods wellknown in the art.

In some embodiments of this aspect, the lipoprotein-capture ligand islinked to a solid support by use of appropriate linkers well known inthe art. Exemplary solid supports include, without limitation,paramagnetic particles, beads, gel matrix material (e.g., agarose,Sephadex®), and the like.

Further to this aspect, in some embodiments the present inventionprovides methods for removing Lp(a) from the sample prior tocentrifugation, which method includes the following steps: (a) forming aprecipitate of Lp(a) by admixing the sample with a precipitant for ApoB-containing lipoproteins under conditions sufficient to causeprecipitation of Lp(a); and (b) isolating the Lp(a) containingprecipitate from the first solution. “Preciptant for Apo B-containinglipoproteins” and like terms refer to compounds known to precipitate ApoB, as well known in the art.

In some embodiments of the present invention contemplating purificationof lipoproteins collected by centrifugation methods provided herein, thepresent invention provides methods for removing Lp(a) from collectedlipoproteins, which methods include the following steps: (a) forming aprecipitate of Lp(a) by admixing the collected lipoproteins with aprecipitant for Apo B-containing lipoproteins under conditionssufficient to cause precipitation of Lp(a); and (b) isolating the Lp(a)containing precipitate from the collected lipoproteins.

Further to methods provided herein for removing Lp(a) from a solutioncontaining lipoprotein, an exemplary precipitant for Apo B is, withoutlimitation, DS in the presence of divalent cation. In some embodiments,the divalent cation is Mg²⁺. It has been observed that inclusion of DSresults in a significantly enhanced recovery of LDL with little effecton recovery of HDL. DS can be mixed with lipoprotein-containing sampleat a concentration in the range of about 0.1 to 50 mg/mL. In someembodiments, the DS concentration is about 0.1, 0.2, 0.5, 1.0, 1.5, 2.0,2.5, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 40.0 or even 50.0mg/mL.

In further embodiments, the invention provides methods for obtainingpurified Lp(a), which methods include the following steps: (a)solubilizing the Lp(a) containing precipitate obtained according to anyof the methods provided herein therefor; (b) admixing the solubilizedLp(a) with a solid-support reagent containing a lectin attached to asolid support under conditions suitable to allow formation of aLp(a)-lectin complex; (c) isolating the Lp(a)-lectin complex; and (d)releasing the Lp(a) from the Lp(a)-lectin complex, thereby providingpurified lipoproteins suitable for example for ion mobility analysis.

Further to this method, the lectin may be selected from the groupconsisting of wheat germ agglutinin (WGA), lima bean agglutinin (LGA),phytohemaglutinin (PHA), and horseshoe crab lectin (HCL). In someembodiments, the lectin is WGA. In some embodiments, the solid supportincludes agarose. Methods of manipulation of such lectins, includingreacting with Lp(a) to form a complex, isolating such a complex, andattaching lectin to a solid support, are well known in the art.

Further to this method, in some embodiments the releasing step includeswashing the Lp(a)-lectin complex with a competitive ligand for thelectin. In some embodiments, the competitive ligand isN-acetylglucosamine (NAG). In some embodiments, the releasing stepincludes disulfide reduction, using a dilsufide reducing agent as knownin the art, to reduce the disulfide linking apo (a) and Apo B, therebyreleasing LDL.

In some embodiments of the present invention contemplating furtherpurification of lipoproteins collected by centrifugation methodsprovided herein, the present invention provides methods for removingLp(a) from collected lipoproteins, which methods include the followingsteps: (a) solubilizing the Lp(a) containing precipitate obtainedaccording to any of the methods provided herein therefor; (b) admixingthe solubilized Lp(a) with gamma globulins and proline; (c)precipitating the admixture by addition of a precipitant; and (d)recovering Lp(a) from the precipitate, thereby providing purifiedlipoproteins suitable for ion mobility analysis. As known in the art,“gamma globulin” refers to the γ-class of immunoglobulins. Exemplaryprecipitants include, without limitation, salts of highly chargedinorganic ions, preferably ammonium sulfate. The concentration of gammaglobulins useful for the present embodiment can be in the range 0.01-0.1ug/mL, 0.1-1.0 ug/mL, 1.0-2.0-ug/mL, 2.0-5.0 ug/mL, 5.0-10.0 ug/mL,10.0-100 ug/mL, 100-1000 ug/mL, or even higher. The concentration ofproline can be in the range 10 uM-100 uM, 100-100-uM, 1-2 mM, 2-5 mM,5-10 mM, or even higher.

Further to methods provided herein contemplating collected lipoprotein,in some embodiments the lipoprotein-containing solution is in contactwith an inert centrifugation matrix. “Inert centrifugation matrix” andlike terms in the context of centrifugal purification methods of thepresent invention refer to materials which do not chemically react withlipoproteins but which nonetheless enhance purification. Without wishingto be bound by any theory, it is believed that the inert centrifugationmatrix acts to stabilize the contents of a centrifugation tube aftercentrifugation such that, for example, artifacts introduced duringdeceleration and/or pipetting of lipoprotein or other fraction from thetube are minimized. Exemplary inert centrifugation matrices include,without limitation, gel slurries or inert beads. In some embodiments,the gel slurry is a Sephadex® gel matrix. In some embodiments, the inertcentrifugation matrix includes inert beads. Exemplary inert beadsinclude, without limitation, glass beads, polystyrene beads, and thelike, adapted to sink to the bottom of the first solution in acentrifuge tube. Inert beads can be of any convenient size, for examplewithout limitation, about 0.1, 0.2, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.3,3.6, 3.9, 4.0 mm, or even smaller or larger.

In some embodiments of the aspect of the present invention directed tomethods for purifying lipoproteins for ion mobility analysis by the useof polyanionic compounds and one or more divalent cations, thepolyanionic compound is selected from the group consisting of DS,amylopectin and polyvinyl sulfate, preferably DS. In some embodiments,the divalent cation is selected from the group consisting of Mg²⁺ andCa²⁺, preferably Mg²⁺.

In some embodiments, the present invention provides methods forpurifying lipoproteins for ion mobility analysis, which methods do notinclude centrifugation. In some embodiments, a solution comprisinglipoproteins and non-lipoproteins is admixed with one or morelipoprotein-capture ligands capable of binding lipoproteins to form alipoprotein/lipoprotein-capture ligand complex. In some embodiments,after formation of a lipoprotein/lipoprotein-capture ligand complex, thecomplex so formed is isolated by methods known in the art including,without limitation, immunomagnetic methods. In some embodiments, afterisolation of a lipoprotein/lipoprotein-capture ligand complex, theliprotein is released from the lipoprotein/lipoprotein-capture ligandcomplex by methods known in the art and described herein.

In view of any of the aspects contemplating isolation and/or purifyingof lipoproteins described herein, in another aspect the inventionprovides methods for analyzing the size distribution of lipoproteins byion mobility analysis. In some embodiments, the one or more lipoproteinsare obtained from a body fluid such as a plasma specimen from anindividual. In some embodiments, the one ore more lipoproteins areselected from the group consisting of HDL, LDL, Lp(a), IDL and VLDL. Insome embodiments, the method further includes the step of using thedetermined lipoprotein size distribution to conduct an assessment of theindividual, the assessment selected from the group consisting oflipid-related health risk, cardiovascular condition, risk ofcardiovascular disease, and responsiveness to a therapeuticintervention.

“Assessment” in the context of lipid-related health risk, cardiovascularcondition, and risk of cardiovascular disease, refers to a statisticalcorrelation of the resulting lipoprotein size distribution withpopulation mortality and risk factors, as well known in the art.Assessment in the context of responsiveness to a therapeuticintervention refers to comparison of the lipoprotein size distributionbefore and after a therapeutic intervention is conducted. Exemplarytherapeutic interventions include, without limitation, theadministration of drugs to an individual for the purpose of loweringserum cholesterol, lowering LDL, IDL, and VLDL, Lp(a) and/or raisingHDL, as known in the art.

In some embodiments, the results of lipoprotein analyses are reported inan analysis report. “Analysis report” refers in the context oflipoprotein and other lipid analyses contemplated by the invention to areport provided, for example to a clinician, other health care provider,epidemiologist, and the like, which report includes the results ofanalysis of a biological specimen, for example a plasma specimen, froman individual. Analysis reports can be presented in printed orelectronic form, or in any form convenient for analysis, review and/orarchiving of the data therein, as known in the art. An analysis reportmay include identifying information about the individual subject of thereport, including without limitation name, address, gender,identification information (e.g., social security number, insurancenumbers), and the like. An analysis report may include biochemicalcharacterization of the lipids in the sample, for example withoutlimitation triglycerides, total cholesterol, LDL cholesterol, and/or HDLcholesterol, and the like, as known in the art and/or described herein.An analysis report may further include characterization of lipoproteins,and references ranges therefore, conducted on samples prepared by themethods provided herein. The term “reference range” and like terms referto concentrations of components of biological samples known in the artto reflect typical normal observed ranges in a population ofindividuals. Exemplary characterization of lipoproteins in an analysisreport may include the concentrations of non-HDL lipoproteins and Lp(a)determined by ion mobility. Further exemplary characterization oflipoproteins, determined for example by ion mobility analyses conductedon samples prepared by methods of the invention, include theconcentration and reference range for VLDL, IDL, Lp(a), LDL and HDL, andsubclasses thereof. An analysis report may further include lipoproteinsize distribution, obtaining for example by ion mobility analysis, of asample prepared by methods of the invention. Entries included in anexemplary analysis report are provided in Example 7.

EXAMPLES Example 1—Comparison of Lipoprotein Purification Using D₂O andLow-Salt Solution

A serum sample (25 uL) was processed either using a low-density saltsolution (1.151 g/mL) (i.e., “low-density salt sample”) or D₂O (200 uLeach). Samples were centrifuged at 223,000×G for 3.7 hr (low-densitysalt sample) or 2 hr (D₂O). Following removal of the top 100 uL aftercentrifugation, the low-density salt sample was dialyzed againstammonium acetate solution and diluted to 1:200 before ion mobilityanalysis. The D₂O sample was diluted directly after centrifugation to1:200 with ammonium acetate prior to ion mobility analysis. Results ofion mobility analysis are presented in FIG. 2.

Example 2—Effect of Purification on Apo A, Apo B, and TC Recovery

To assess whether HDL (Apo A1) was preferentially lost in proceduresemploying D₂O, three samples as shown in FIG. 3 (i.e., 749, 1043, 14:arbitrary and unique patient identification numbers) were subjected tolipoprotein isolation employing D₂O together with RGD/DS solution(7.5/2.5 mg/mL, respectively) to remove albumin. Samples were eachprepared in replicates of six. The isolated individual top 100 uL wereeach analyzed for content of Apo A1 (HDL), Apo B (LDL, IDL, VLDL) andtotal cholesterol (TC). Plasma or serum apolipoproteins AI and B weremeasured by standardized ELISA using commercially available monoclonalcapture antibodies (Biodesign International, Saco, Minn.) and anti-humangoat polyclonal detection antibodies, purified and biotinylated,(International Immunology Corp., Murrieta, Calif.) in a non-competitivesandwich-style immunoassay. Concentration was measured by addition ofstreptavidin conjugated peroxidase followed by color development usingortho-phenyline-diamine. Lipoprotein calibrators were standardized usingCDC #1883 serum reference material (Center for Disease Control, Atlanta,Ga.) and pooled reference sera (Northwest Lipid Research Clinic,Seattle, Wash.). Total cholesterol was measured using commerciallyavailable assay kit reagents (Bayer Health Care, Tarrytown, N.Y.)according to manufacturers instructions and modified for analysis of 25μl serum or plasma plus 200 μl cholesterol reagent per microtiter platewell. Standards, controls, samples and reagent background were measuredafter color development using a microtiter plate reader. The results(FIG. 3) show the mean recovery of each sample compared to the totalpresent in each serum. Without wishing to be bound by any theory, thepurification procedure did not result in preferential loss of HDL, asjudged by equivalent recovery of Apo A1 and Apo B.

Example 3—Effect of Varying RGD on Lipoprotein Fraction Recovery

A serum sample was mixed with varying amounts of RGD (10, 15, 20, 25mg/mL) and incubated on ice for 15 min before being overlaid on acushion of D₂O. After centrifuging for 120 min at 223,000×G, the top 100uL was removed and diluted 1:200 with ammonium acetate solution. Sampleswere then analyzed by ion mobility analysis. Results are shown in FIG.4.

Example 4—Purification of Lipoproteins Employing RG 19, DS, AA

With reference to FIG. 5, in order to assess the effect of DS on theremoval of albumin and recovery of lipoproteins in both theextraction/purification and diluent, a serum sample (5 uL) was extractedwith 20 uL of 7.5 mg/mL RGD alone (legend “C/D” in FIG. 5) or 20 uL of acombination of 7.5 mg/mL RGD and 2.5 mg/mL DS (legend “A/B” in FIG. 5).The DS molecular weight used for both the extraction and diluent is 10K.After 15 min Incubation on ice each sample was centrifuged for 2 hr 15min at 223,000×G at 10 C. The top 100 uL was removed and diluted 1:200with either 25 mM ammonium acetate solution (legend “B/D” in FIG. 5) or25 mM ammonium acetate containing 5 ug/mL DS (legend “A/C” in FIG. 5).

Example 5—Result of Purification of Lipoproteins Employing DS in Diluent

With reference to FIG. 6, a lipoprotein-containing serum sample preparedby a 18 hr density separation, using methods well known in the art, wasemployed after dialysis to assess the effect of DS in the diluent on therecovery of LDL. An aliquot of the centrifuged serum sample was diluted1:200 with 25 mM ammonium acetate in the absence of DS and subjected toion mobility analysis. Another aliquot was diluted 1:200 with 25 mMammonium acetate in the presence of 5 ug/mL DS. Duplicate runs of eachsample are shown in FIG. 6.

Example 6—Purification of Lipoproteins Employing RG 19, DS, D₂O

Lipoprotein-containing samples obtained from plasma were mixed brieflyby vortexing. Five uL of sample, or optionally control, were mixed with20 uL of an albumin removal reagent containing 7.5 mg/mL RGD (Sigma),2.5 mg/mL DS (Sigma) and 0.5 mg/mL EDTA (Spectrum Chemicals) andincubated on ice for 15 min. Following incubation the sample mixture wasoverlaid on D₂O (Medical Isotopes) 200 uL in a Ti 42.2 ultracentrifugetube (Beckmann). The samples were then ultracentrifuged at 10° C. for135 min at 223,000×g (42,000 rpm). Following ultracentrifugation thelipid fraction (85 μL) was removed from the top of the centrifuge tube.Prior to analysis by ion mobility, the samples were diluted to a finaldilution of 1:800 in 25 mM ammonium acetate 0.5 mM ammonium hydroxide pH7.4 for HDL analysis. For LDL analysis samples were diluted 1:200 in thesame diluant containing 5 ug/mL DS. Final dilutions were made in deepwell 96 well plates and placed in an autosampler with the cool stackmaintained at 6° C., prior to ion mobility analysis.

Example 7—Result of Purification and Analysis of Lipoproteins in SerumSamples

Serum was separated from whole blood collected via venipuncture.Following separation the serum was divided into three portions, onealiquot analyzed for HDL, triglycerides, and total cholesterol contentusing traditional methods well known in the art. LDL is calculated fromthese results. In preferred embodiments, if triglycerides are greaterthan 400 mg/dL then LDL is measured directly. The second aliquot wasanalyzed for its Lp(a) content using an immunoassay, well known in theart. Ion mobility analysis was employed for the third aliquot tofractionate the lipoproteins.

In a typical production procedure, sample(s) together with controls, onesample known to be LDL pattern A (control A) and one sample known to bepattern B (control B) as known in the art, are placed on the PerkinElmer JANUS multiprobe. 30 uL of controls and sample(s) are transferredto a separate tube and mixed, and 120 uL of the RG19 dextran, DS, EDTAsolution is added. The tubes are then transferred to ice for a 15-minuteincubation. Following the 15 min incubation the tubes are returned tothe multiprobe. In the meantime, centrifuge tubes have had two 4 mmbeads added to them, and these are then placed on the multiprobe where120 uL of D₂O is added to each centrifuge tube. Controls and sample(s)are then overlaid on the D₂O by the multiprobe before being transferredto the ultracentrifuge rotor (Ti 42.2). Samples are then spun for 135min at 10 C at 223,000×G (42,000 rpm). Following centrifugation, thecentrifuge tubes are removed carefully and placed on the multiprobewhere the top 85 uL (+/−5 uL) is removed to a separate tube. Once allsamples are collected the multiprobe makes two dilutions for eachcontrol and sample. One dilution is a final dilution of 1:200 withammonium acetate solution containing 5 ug/mL DS; the second is a 1:800dilution with just ammonium acetate solution. The two dilutions are thenrun on the ion mobility instrument. Following analysis the particlenumbers are converted to nmol/L using conversions well known in the art.The data from the HDL run (1:800) and the larger lipoproteins (1:200)are combined and reported together with the biochemical data fromaliquots 1 and 2. The profile of the lipoproteins is also reported aswell as the total LDL particle concentration and the LDL peak particlesize, which is used to determine the LDL phenotype. An exemplaryassessment report resulting from combining these data is provided inTable 2 (numerical representation) and FIG. 7 (graphical representationof lipoprotein profile).

TABLE 2 Lipoprotein Fraction by Ion Mobility Assay In Out of ReferenceComponent Range Range Units Range Lipid Panel Cholesterol, Total 328(High)* mg/dL <200 LDL Cholesterol 249 (High) mg/dL <130 HDL Cholesterol62 mg/dL  >50 VLDL Cholesterol 17 mg/dL  <30 Triglycerides 85 mg/dL <150Non-HDL Cholesterol 266 (High) mg/dL <160 Lipoprotein (a) 25 nmol/L  <75LDL Particle Profile LDL Particles, Total 886 nmol/L 272-1181 LDLParticle size 228.1 Ang 215.4-232.9  LDL Phenotype A Type** ALipoprotein Particles LDL I large 226 (High) nmol/L 51-186 LDL II large426 nmol/L 91-574 LDL III small 187 nmol/L 82-442 LDL IV small 47 nmol/L33-129 HDL 2b large 1425 nmol/L 384-1616 HDL 2a intermediate 5616 (High)nmol/L 903-3779 HDL 3 small 9229 (High) nmol/L 475-4244 IDL 1 large 39(High) nmol/L 10-38  IDL 2 small 66 (High) nmol/L 11-48  VLDL large 1.9(High) nmol/L 0.2-1.8  VLDL intermediate 6.4 (High) nmol/L 1.0-5.7  VLDLsmall 25.1 nmol/L 5.8-26.6 *“High” and “Low” refer to above or belowrange, respectively. **“Type” refer to phenotype as determined byparticle size with cutoff approximately at LDL II (215.4 A), as known inthe art.

To obtain more accurate lipoprotein profile using differential ionmobility as discussed above, one may adjust the results for any loss oflipoprotein during handling (e.g. sample centrifugation, pipeting anddilutions) prior to the ion mobility apparatus. This may be achieved byadding one more types of labeled lipoproteins to a sample as an internalstandard. By following the label during processing, the recovery of thelabeled lipoprotein can be used to adjust upwards the concentration ofthe same but unlabeled lipoprotein present in the original sample. Forexample, an aliquot of the lipoprotein isolate after centrifugation ismeasured for fluorescent signal and compared with an aliquot directlyfrom the starting stock sample (not centrifuged). The difference insignal represents the proportion of unknown sample recovered, and allowsa more accurate calculation of lipoprotein concentration in plasma orserum.

The following method was used to conjugate a fluorescent molecule to HDLsubfractions. This method may be applied to other types of lipoproteins.HDL was isolated from plasma by sequential flotation to obtainlipoproteins within density interval 1.063-1.20 g/mL. The total HDLfraction was then dialyzed to salt background density 1.184 g/mL andcentrifuged for 28 hrs at 40,000 rpm, 10° C. in a fixed angle 50.3Beckman rotor. The 6 ml centrifuge tube was then pipetted to obtainpredominantly large, intermediate and small HDL subfractions, T[0-1],T[1-3] and T[3-6], respectively. The subfractions were then dialyzedagainst 100 mM NaHCO₃, pH 8.5, 4° C. overnight. Protein concentrationwas measured in each subfraction using the Lowry method.

HDL subfractions were then labeled with fluorescent probe AlexaFluor 488(carboxylic acid, succinimidyl ester ‘mixed isomers’, Molecular ProbesCat #A-20000, Mol. Wt. 643.42, Abs A494 nm/Em 517 nm) according tomanufacturer's instructions. Briefly, HDL subfractions were combinedwith AF488 at a suggested optimal ratio 10:1 (wt:wt) maintaining optimalconcentrations of HDL and AF488, >2 mg/ml and 10 mg/ml, respectively.The protocol and quantities of the solutions used are listed below.

Incubation Mixtures:

Stk Stk Stop Co Ligand Co AF488 Tot. Vol Soln mg/ml μl mg mg/ml μl mg μlμl HDL Subfr. 3.59 560 2.01 10 20.104 0.2010 580 40 T[0-1] T[1-3] 3.18625 1.99 10 19.875 0.1988 645 40 T[3-6] 6.39 785 5.02 10 50.162 0.5016835 100 Total 90.1405 0.901405

-   -   1—Add HDL subfr to glass vial with mag-stir bar    -   2—While stirring at rm. temp., add AF488 volume to ligand        slowly.    -   3—Incubate mixture for 1 hour w/continuous stirring.    -   4—Add Stop Soln (1.5 M Tris, pH 8.0). Incubate at rm temp 30        min.    -   5—Dialyze labeled HDL Subfrs to 20 mM Tris. 150 mM NaCl, 0.27 mM        EDTA, pH8 [in cold box, protect from light] vs. 1 liter        overnite, and 2×1 L dialysate volume changes.

The AF488 labeled HDL subfractions were then tested for signalsensitivity at various dilutions in buffer from 250 to >30000. Thelabeled HDL subfractions were also tested for signal sensitivity whendiluted in various plasma preparations before and after centrifugationfor isolation of lipoproteins.

Additional dilution and sensitivity tests were performed after a secondcentrifugal isolation of the labeled HDL subfractions at density <1.23g/mL to remove unconjugated fluorescent label from the HDL:AF488conjugates.

The fluorescent probe fluorescein-5-EX, succinimidyl ester, obtainedfrom Molecular Probes (Cat #F-6130), was used to label HDL subfractionsin the same manner as described above for AF488. The above methods werealso used to fluoresencently label VLDL and LDL. Additional tests wereconducted to fluoresence label combined high molecular weight standards(Pharmacia HMW Standard Mix) containing thyroglobulin, apoferritin,catalase, lactate dehydrogenase, and albumin.

In embodiments of aspects of the present invention contemplatinganalysis and/or display of lipoprotein distributions, as exemplifiedwithout limitation by FIG. 1, FIG. 7 and the like, the ion mobilitydata, obtained with a differential ion mobility analyzer, can beprocessed prior to presentation for clinical interpretation. Unlessotherwise specified, “differential ion mobility data” and like terms inthe context of raw data from an ion mobility analysis, or processed datafor presentation for clinical interpretation, refer to differential ionmobility particle size distributions having an independent variablecorrelated to the diameter of a particle, and an observed dependentvariable correlated with particle count. In some embodiments, theindependent variable is voltage or the corresponding electrical fieldgenerated by the voltage (see Eqn. 3). In some embodiments, theindependent variable is particle diameter. In some embodiments, thedependent variable is particle count. In some embodiments, the dependentvariable is the number of particles counted during a specified timeperiod, for example without limitation 0.001-0.01, 0.01-0.1, 0.1-1, 1-2s or even longer. In some embodiments, the specified time period is 0.1s.

“Processing of the differential ion mobility data” and like terms referto manipulations of data, which manipulations, when taken in total, mayprovide graphical and/or numeric results which accurately andreproducibly reflect the lipoprotein distribution, and/or concentrationsof individual lipoprotein classes and subclasses thereof, within asample. Exemplary manipulations useful in the processing of differentialion mobility particle size distribution data include, withoutlimitation, multiplication by a constant, convolution with a function,addition and/or subtraction of a constant numeric value or a functionincluding without limitation correction for the contribution of acontaminant, numeric integration, smoothing, and other arithmeticmanipulations known in the art. Accordingly, processing of thedifferential ion mobility particle size distribution data can beemployed for a variety of reasons, including without limitation,correction to accurately reflect physiological concentrations oflipoproteins in a sample, scaling to correct for specific instrument andprocess efficiencies, removal of data representing contributions of acontaminant, and the like. “Specific instrument and processefficiencies” and like terms refer to the detection and correction forchanges in analyte (e.g., lipoprotein) concentration during processingand analysis. Exemplary specific instrument efficiencies include anapparent dilution introduced during electrospray wherein formation ofthe Taylor cone results in an apparent dilution of lipoprotein in theresulting particle-laden gas stream. Efficiencies are measured bymethods employing instruments having quantified efficiencies well knownto practitioners in the art. “Contribution of a contaminant” and liketerms in the context of differential ion mobility particle sizedistribution data refer to data, for example without limitation particlecount from a differential ion mobility instrument analysis, resultingfrom non-lipoprotein species counted in the differential ion mobilityinstrument and included in the differential ion mobility particle sizedistribution data obtained therefrom. Exemplary non-lipoprotein speciesin this context include, without limitation, any reagent disclosedherein and albumin in monomeric and/or multimeric form.

In some embodiments, the contribution to the ion mobility particle sizedistribution data due to a reagent described herein is subtracted fromthe ion mobility size distribution data during processing of the data.For example, without wishing to be bound by any theory, it is believedthat a contribution due to RGD in particle size distribution data from adifferential ion mobility analysis (i.e., differential ion mobilityparticle size distribution data having particle count versus particlediameter) can be represented by a one or more decaying exponentialfunctions over selected diameter regions. Accordingly, in someembodiments the differential ion mobility particle size distributiondata are fit in a selected region to a function having the form of Eqn4:y ₁ =k ₁ *e ^((−0.7*d))  (4)wherein y₁ is the best fit for the contribution to the ion mobility asdetermined by methods well known in the art, k₁ is an empirical constantof the fit, and d is the particle diameter. The above Eqn. 4 is validfor particle diameters of greater than 2 nm. In some embodiments, theregion of the fit is 3-6, 3-4, 3-5, 3-6, 4-6, or 5-6 nm (particlediameter), preferably 3-4 nm. In some embodiments, the entire set of ionmobility data is corrected by the function resulting from a fit to Eqn.4.

In some embodiments, the differential ion mobility particle sizedistribution data are further processed to account for a contributiondue to albumin inclusion in the sample taken for ion mobility analysis.In some embodiments, the construction of a correction for albumin (i.e.,“albumin correction curve”) is initially afforded by a piecewisefunction having the following form:

Dependent variable Region region, nm Functional correction 1   0 <= d <7 0 2   7 <= d < 7.1 y₂ = k₂ * _(e)(^(−2.56) * ^(7.1)) Eqn. (5) 3 7.1 <=d < 8.5 y₃ = k₃ * _(e)(^(−2.56) * ^(d)) Eqn. (6) 4 8.5 <= d < 15Empirical (from spiked albumin data)wherein y₂ and y₃ are the functional values in regions 2 and 3,respectively, k₂ and k₃ are empirical constants determined by methodswell known in the art, and d is the particle diameter. “Empirical (fromspiked albumin data)” refers to the effect on ion mobility particle sizedistribution data of subtracting from the distribution an amount ofalbumin equivalent to the amount of albumin in the measureddistribution.

In some embodiments, the albumin correction curve is further modified toaccount for the presence of albumin dimer. It has been found empiricallythat albumin dimer is typically present in samples for ion mobilityanalysis in the range 1-10%, 1-8%, 2-8%, 2-7%, 2-6%, 2-5%, preferably2%, and that an albumin correction curve can be scaled in a particularregion to account for, and to gradually suppress, the presence ofalbumin dimer. In some embodiments, the lower diameter limit of thisparticular region is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 nm.In some embodiments, the upper diameter limit of this particular regionis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or even 15 nm. In someembodiments, the range of this particular region is 0-15, 5-10, 7-9,preferably 7.9-8.4 nm. Accordingly, the albumin correction curve can bemodified by a function having the form of Eqn 7:y′=y*((d−lowerlimit)*2*dimer+(upperlimit−d)*2)  (7)wherein y is the albumin correction curve, y′ is the albumin correctioncurve after gradual suppression of the presence of albumin dimer, d isthe particle diameter, lowerlimit and upperlimit are the lower and uppersize limits for the correction, respectively, and dimer is the selectedpercentage dimer concentration. In some embodiments, the region in whichthe presence of dimer is suppressed is between 7.9 nm (lowerlimit) and8.4 nm (upperlimit).

In some embodiments, a theoretical curve representing albumin monomer isfit to the ion mobility particle size distribution of a sample in aparticular region, using curvefitting methods well known in the art. Insome embodiments, this theoretical curve is represented by a functionhaving the form of Eqn. 8:

$\begin{matrix}{y_{m} = {k_{m}*e^{({{- k_{a}}*d})}}} & (8)\end{matrix}$wherein y_(m) is the theoretical number distribution of albumin monomer,k_(m) and k_(a) are empirically derived constants In some embodiments,k_(a) is in the range 0.1-10, 1-5, 2-4 or 2-3. In some embodiments,k_(a) is 2.56. In some embodiments, this particular region is the range0-15, 5-10, 6-9, 7-8, preferably 7.3-7.5 nm. In some embodiments, afterdetermination of the contribution of albumin monomer which results inthe best fit to Eqn. 8, the ion mobility particle size distribution dataare corrected by subtracting Eqn. 7 therefrom, scaled by the samecontribution. In some embodiments, the correction afforded bysubtracting Eqn. 7 is conducted in a particular region, for examplewithout limitation, 0-15, 2-12, 4-10, preferably 6-10 nm. In someembodiments wherein the correction does not contemplate the range 10-11nm, a corresponding correction in the region 10-11 nm is conducted bymultiplying Eqn 7. by a factor of (11-diameter) and subtracting theresult from the ion mobility particle size distribution data.

The process described above may be implemented in a variety ofelectronic devices, such as desktop or laptop computers or handhelddevices, for example. Such devices are well known to those skilled inthe art. Additionally, the results may be displayed on a monitor,printed or stored on a memory device, such as a hard drive, CD ROM, CDR/W, flash memory or the like. Further, the results may be madeavailable to other devices through a network, which may be a privatenetwork or a public network, such as the Internet. In this regard, theelectronic device and/or the memory device may be accessible through thenetwork.

In one embodiment, the measured values are compared to empiricallydetermined ranges to perform a diagnosis based on a patient's serum orplasma values falling within or outside a range. The table belowillustrates one exemplary set of ranges for such a diagnosis:

nmol/L HDL 3 HDL 2a HDL 2b Female 475-4224  903-3779 384-1616 Male613-3344 1174-3744 169-1153

nmol/L LDL IV LDL III LDL II LDL I LDL Total Female 33-129  82-442 91-574 51-186 272-1189 Male 38-164 136-627 200-596 48-164 508-1279

LDL Paricle Size (A) Female 215.4-232.9 Male 212.3-230.9

nmol/L IDL 2 IDL 1 Female 11-48 10-38 Male 12-59 11-41

nmol/L VLDL sm VLDL int VLDL lg Female 5.8-26.6 1.0-5.7 0.2-1.8 Male5.0-23.0 1.1-7.3 0.2-2.5

Ion mobility spectrometry provides a way to measure the sizedistribution of nanoparticles based on gas-phase particle electricalmobility. This methodology was adapted for measuring the sizedistribution of lipoprotein particles. The method was automated andgenerated profiles of particle number and particle mass versus particlediameter in about one minute. Lipoproteins are first enriched (plasmaprotein removal) by ultracentrifugation and then diluted in a volatilebuffer and electrosprayed. A charge neutralization process leaves awell-characterized fraction of the particles with a single charge. Thecharged particles are drawn through a Differential Mobility Analyzer(DMA), which allows particles of a narrow size to pass to a particlecounter as a function of a voltage applied to the DMA. By scanning theapplied voltage, particle number distributions are obtained for HDL,LDL, IDL and VLDL. The measurements are based on first principles and donot need to be calibrated with respect to particle size. Particle numberdistributions are converted into particle mass distributions. Using thismethod, the intra-assay variation for LDL diameter was <0.6%, forconcentration, <10% for HDL and LDL and <15% for IDL and VLDL. Theinter-assay reproducibility was <1.0% for LDL particle size, and forconcentration, <15% for HDL and LDL and <20% for IDL and <25% for VLDL.The table below shows the summary data, expressed as mean and SD, usedto generate reference ranges for the individual lipoprotein fractions. Atotal of 259 healthy individuals (191 F, 68 M) who met the current NCEPATP III criteria for optimal lipid/lipoprotein levels: total cholesterol(chol)<200, LDL chol<100, HDL chol>40 (M)>50 (F), triglyceride<150 mg/dLwere used in the study. The results show the expected difference betweengenders, males having higher concentrations of smaller LDL particles andfemales having increased HDL 2b.

P Lipoprotein Mean SD males vs. Fraction Gender nmol/L nmol/L femalesHDL 3 Female 1443 847 0.071 Male 1646 602 HDL 2b Female 834 299 <0.003Male 494 258 HDL 2a Female 2343 719 0.850 Male 2325 655 LDL IV Female 7024 <0.003 Male 84 31 LDL III Female 212 103 <0.003 Male 313 125 LDL IIFemale 336 125 <0.003 Male 407 119 LDL I Female 112 35 0.012 Male 100 29Total LDL Female 727 227 <0.003 Male 893 193 Angstrom Angstrom LDL PeakFemale 225.7 4.48 <0.003 Diameter Male 221.7 4.19 IDL 1 Female 16 90.003 Male 20 11 IDL 2 Female 25 10 0.007 Male 29 12 VLDL small Female11.2 5.7 0.506 Male 10.7 4.8 VLDL inter Female 3.0 1.3 <0.003 Male 3.71.6 VLDL large Female 0.9 0.4 <0.003 Male 1.2 0.6

Ranges for the remainder of the population (abnormal) with one or morecriterion outside the ATP III guidelines were determined. These showedexpected differences with increased concentrations of smaller LDL aswell as decreased size and decreased concentration of HDL 2b (B) withlittle change in HDL 2a and 3.

The methods described above may be carried out in a variety ofapparatuses. For example, U.S. Patent Publication No. 2003/0136680 toBenner et al. describes an apparatus by which a sample solution in acentrifuging tube is expelled through a capillary tube where it becomesionized by the electrospray process as it exits the capillary tube.Thus, the pressure differential caused by the pressure chamber transfersthe ionized sample into a gas stream, which then carries the sample to amobility analyzer. Once the sample in the centrifuging tube is analyzed,another tube of sample is placed within the pressure chamber. In thisarrangement, however, since only a small volume of the sample isprovided at any time in a centrifuging tube, the flow rate of the samplethrough the capillary can vary substantially over time even if thepressure in the pressure chamber is maintained, thereby affecting thequantitative determinations from the ion mobility analyzer based onpredicted flow rates.

Embodiments of the present invention address these concerns. Inaccordance with embodiments of the invention, a constant flow rate isachieved by pumping the sample through a capillary and by ionizing (orcharging) the sample within the capillary during flow to the ionmobility analyzer. FIG. 8 illustrates an exemplary apparatus for ionmobility analysis according to an embodiment of the invention. The ionmobility analysis apparatus 10 of FIG. 8 includes an ion mobilityanalyzer 20 similar to that illustrated in U.S. Patent Publication No.2003/0136680. The ion mobility analyzer 20 is capable of countingparticles flowing therethrough. The ion mobility analyzer 20 may beprovided with an electronic device (not shown), such as a computer,capable of processing the data in accordance with, for example, thealgorithm described above.

A charged particle stream of the sample is provided to the ion mobilityanalyzer 20 from an autosampler 22. The autosampler 22 may be a roboticsystem for automatically supplying a sample. One such autosampler ismodel HTC PAL, Leap Technologies of Carrboro, N.C. In one embodiment,the autosampler is a robotic device that only supplies purified samplefrom a rack of tubes or from a multiwell plate to the pump(s). Theautosampler 22 can provide a substantially continuous supply of samplesfor ion mobility analysis without the need for substantial humanintervention.

Sample from the autosampler 22 is supplied to a first pump 26 through aninjection port 24. In this regard, the autosampler 22 may include areservoir (not shown) in which the purified sample is contained. Theinjection port 24 may be a part of the first pump 26. The first pump 26is a high flow-rate (or high-flow) pump capable of pumping the samplefrom the autosampler 22 at a relatively high flow rate (e.g., greaterthan or equal to 1.0 microliter per minute). In one embodiment, thehigh-flow pump pumps the sample from the autosampler 22 at a rate ofapproximately 5-20 microliters per minute. Most preferably, thehigh-flow pump pumps the sample at a rate of approximately 10microliters per minute. Suitable high flow pumps are obtained fromEksigent Technologies, 2021 Las Positas Ct Suite 161, Livermore, Calif.

From the first pump 26, the sample is supplied to a second pump 30. Thesecond pump 30 is a low flow-rate (or nanoflow) pump capable of pumpingthe sample to a capillary 34 at a relatively low rate (e.g., less thanor equal to 1.0 microliters per minute) to enable proper ionization orcharging of the particles of the sample, as described below. In oneembodiment, the nanoflow pump pumps the sample to the capillary at arate of approximately 100-200 nanoliters per minute. Most preferably,the nanoflow pump pumps the sample at a rate of approximately 200nanoliters per minute. Suitable nanoflow pumps are obtained fromEksigent Technologies, 2021 Las Positas Ct Suite 161, Livermore, Calif.

In one embodiment, a combination pump assembly may be used in place ofthe two pumps. For example, a pump assembly may include a high-flowcomponent and one or more nanoflow components. An exemplary combinationpump assembly is NanoLC 1-D, available from Eksigent Technologies, 2021Las Positas Ct Suite 161, Livermore, Calif.

In one embodiment, the first pump 26 may supply the sample to aplurality of nanoflow pumps through either a single valve 28 or aplurality of valves.

Flow to and through the capillary 34 may be controlled via a valve 32,which may be part of the second pump 30 or may be a separate valvepositioned within the capillary 34. The valve 32 ensures a constant flowrate of the sample through the capillary 34 downstream of the valve 32.The valve 32 may be electronically controlled to maintain the constantflow rate. In this regard, the valve may be controlled in response tosensors or meters positioned downstream of the valve 32.

The sample particles are charged during their flow through the capillary34 by an ionizer 40. As will be understood by those skilled in the art,the actual ionization or charging of the particles may occur as theparticles exit the capillary into the ion mobility analyzer. In oneembodiment, the ionizer 40 is a conductive union assembly positionedaround a portion of the capillary 34. Conductive unions (also known as aconductive junctions) apply an electrical current around a very thinflow to provide an electrical charge to the flow. One exemplaryconductive union assembly is described in U.S. Pat. No. 7,075,066, whichis incorporated herein by reference in its entirety. The charged sampleparticles are then supplied through the capillary 34 to the ion mobilityanalyzer 20.

FIGS. 9A and 9B illustrate exemplary embodiments of the conductive unionfor use in charging the flow of the sample particles through thecapillary. Referring first to FIG. 9A, a conductive union assembly 40 ais formed around the capillary 34. An ionization region 35 of thecapillary 34 is surrounded by a conductive union 42. A voltage appliedto the conductive union 42 causes charging of the particles in the flowthrough the ionization region 35 of the capillary 34. For a detailedexplanation of the operation of the conductive union assembly 40 a,reference may be made to U.S. Pat. No. 7,075,066.

Referring now to FIG. 9B, another embodiment of a conductive unionassembly is illustrated. In the embodiment of FIG. 9B, a conductiveunion assembly 40 b forms a microtite region 37 in a portion of thecapillary 34 through which the sample flows. The microtite region 37 mayform a joint, or a seal, between two sections of the capillary. Themicrotite region 37 has a small dead volume in which the sampleparticles are charged. In one embodiment, the microtite region 37 has adead volume of approximately 5-50 nanoliters. In a most preferredembodiment, the microtite region 37 has a dead volume of approximately10-15 nanoliters. The microtite region 37 is preferably formed ofstainless steel. The conductive union assembly 40 b includes aconductive union 44 formed around the microtite region 37. A voltageapplied to the conductive union 44 causes charging of the particles inthe flow through the microtite region 37.

Thus, the ion mobility analyzer 20 is provided with a controllednanoflow of the sample at a substantially time-invariant rate. In thisregard, the flow rate preferably varies by less than five percent from anominal rate, more preferably by less than two percent and, mostpreferably by less than one percent. This allows for a more consistentand reliable analysis to be performed by the ion mobility analyzer 20.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses which will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof and” consisting of may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any two different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

What is claimed is:
 1. A system for analyzing size distribution oflipoproteins, the system comprising a processor and memory havingmachine instructions stored thereon, the instructions being executableby the processor to cause the processor to: collect, via an ion mobilityanalyzer configured to receive samples for differential charged-particlemobility analysis, differential ion mobility data for a sample havingone or more lipoproteins; determine, using the differential ion mobilitydata, a lipoprotein particle size distribution for one or morelipoproteins, wherein determining the lipoprotein particle sizedistribution comprises determining a best fit of the differential ionmobility data, for a set of one or more regions of interest forlipoprotein particle sizes, to one or more decaying exponentialfunctions over the regions of interest; generate a visual representationof the lipoprotein particle size distribution; and output the visualrepresentation via a display or a printer.
 2. The system of claim 1,wherein the instructions are executable to cause the processor togenerate a graphical representation of the lipoprotein particle sizedistribution as the visual representation.
 3. The system of claim 2,wherein the instructions are further executable to cause the processorto determine a curve corresponding to the best fit, and include thecurve in the visual representation.
 4. The system of claim 1, whereinthe sample on which the differential ion mobility data is based furtherincludes a non-lipoprotein, and wherein the instructions are furtherexecutable to cause the processor, in determining the lipoproteinparticle size distribution, to determine a differential mobilityparticle size distribution for the sample, and subtract a contributionof the non-lipoprotein from the differential mobility particle sizedistribution to obtain the lipoprotein particle size distribution. 5.The system of claim 1, wherein the differential ion mobility data isbased on a sample that is obtained by: a) Admixing a solution comprisinglipoproteins and non-lipoproteins with one or more polyanionic compoundsand one or more divalent cations; b) Allowing a precipitate containinglipoproteins to form in said admixed solution; and c) After step b),collecting the precipitated lipoproteins.
 6. The system of claim 1,wherein the best fit is of the form:y ₁ =k ₁ *e ^((−0.7*d)); where y₁ is a contribution to the lipoproteinparticle size distribution, k₁ is an empirical constant of the best fit,and d is particle diameter; wherein the determining the best fitincludes calculating a value for k₁.
 7. A method of analyzing sizedistribution of lipoproteins, the method comprising: providing, via anautosampler, a charged particle stream of a sample to an ion mobilityanalyzer, the sample including lipoproteins; performing, via the ionmobility analyzer, differential charged-particle mobility analysis ofthe charged particle stream of the sample to generate differential ionmobility data; determining, based on the differential ion mobility data,a lipoprotein particle size distribution for one or more lipoproteins,wherein determining the lipoprotein particle size distribution comprisesdetermining a best fit of the differential ion mobility data, for one ormore regions of interest for lipoprotein particle sizes, to one or moredecaying exponential functions over the regions of interest; andoutputting the lipoprotein size distribution to at least one of adisplay, a printer, and a memory.
 8. The method of claim 7, wherein thesample further includes a non-lipoprotein, and wherein determining thelipoprotein particle size distribution comprises determining adifferential mobility particle size distribution for the sample, andsubtracting a contribution of the non-lipoprotein from the differentialmobility particle size distribution to obtain the lipoprotein particlesize distribution.
 9. The method of claim 8, wherein the differentialmobility particle size distribution is determined for the one or moreregions of particle sizes or a subset thereof.
 10. The method of claim7, further comprising generating a visual representation of thelipoprotein particle size distribution.
 11. The method of claim 7,further comprising generating a graphical representation of thelipoprotein particle size distribution.
 12. The method of claim 11,further comprising determining a curve corresponding to the best fit,and including the curve in the graphical representation.
 13. The methodof claim 12, wherein the curve corresponding to the best fit isdetermined at least in the one or more regions of particle sizes. 14.The method of claim 7, further comprising: a) Admixing a solutioncomprising lipoproteins and non-lipoproteins with one or morepolyanionic compounds and one or more divalent cations; b) Allowing aprecipitate containing lipoproteins to form in said admixed solution; c)After step b), collecting the precipitated lipoproteins; and d)subjecting said precipitated lipoproteins to the differential chargedparticle mobility analysis.
 15. The method of claim 14, wherein themethod does not include centrifugation.
 16. A non-transitory computerreadable medium having machine instructions stored thereon, theinstructions being executable by a processor of a computing device tocause the processor to: receive differential ion mobility data generatedby an ion mobility analyzer, the differential ion mobility data beingbased on differential charged-particle mobility analysis of a samplereceived by the ion mobility analyzer, the sample including one or morelipoproteins; produce, using the differential ion mobility data, alipoprotein particle size distribution in one or more regions ofinterest for lipoprotein particle sizes for one or more lipoproteins,wherein producing the lipoprotein particle size distribution comprisesdetermining a best fit of the differential ion mobility data to one ormore decaying exponential functions over the one or more regions ofinterest; generate a visual representation of the lipoprotein particlesize distribution; and output the visual representation via a display ora printer.
 17. The medium of claim 16, wherein the instructions furthercause the processor to generate a curve fitting the lipoprotein particlesize distribution in the one or more regions of interest or a subset ofthe one or more regions of interest to one or more decaying exponentialfunctions over the regions of interest.
 18. The medium of claim 17,wherein the visual representation is a graphical representation, andwherein the instructions further cause the processor to include thecurve in the graphical representation.
 19. The medium of claim 16,wherein the instructions further cause the processor to generate a curvecorresponding to the best fit in the one or more regions of interest ora subset thereof, and include the curve in the visual representation.20. The medium of claim 16, wherein the sample on which the differentialion mobility data is based includes a non-lipoprotein, and wherein theinstructions further cause the processor to determine a differentialmobility particle size distribution using the differential ion mobilitydata, and subtract a contribution of the non-lipoprotein from thedifferential mobility particle size distribution to obtain thelipoprotein particle size distribution.